PCR is a molecular biological method that is capable of amplifying a target DNA exponentially. Any part of DNA can be amplified once its sequence is identified. PCR was first proposed by K. Mullis in mid-1980s. Since then, PCR has been widely used in biological research fields including molecular genetics which studies genes. PCR exploits the DNA replication activity of DNA polymerase. DNA polymerase facilitates the synthesis of complementary DNA molecule by using single stranded DNA molecule as a template. This single stranded DNA molecule can be obtained simply by boiling a double stranded DNA molecule. This procedure is called ‘DNA denaturation’. In order for DNA polymerase to start DNA synthesis, start site has to be double stranded DNA form. So, to form double stranded DNA, small DNA fragments capable of binding complementarily to both ends of a template DNA should be added in PCR. This complementary binding between DNA fragments and a template DNA is annealing. Only after annealing, DNA synthesis by DNA polymerase can be started. The complementary DNA fragments capable of binding to both ends of a target DNA sequence to be amplified are called oligonucleotide primer or simply primer. After binding of the primer to the template DNA, DNA synthesis extends to the other end by DNA polymerase. PCR cycle is generally composed of the following steps:
1) Denaturation which changes double-stranded template DNA molecule into single stranded DNA molecule;
2) Annealing of the primer to the single stranded DNA template; and
3) Elongation which synthesizes a DNA molecule complementary to the template DNA by DNA polymerase.
After completion of the first PCR cycle, the original template DNA and the PCR product are both used as DNA templates in the subsequent PCR cycle. So, as PCR cycle is repeated, the number of DNA templates is increasing. In an idealized case, the number of existing DNA molecules in a PCR is 2n after n cycles. As a result, (2n−1) copies of the original template DNA are synthesized. In PCR cycles, the first step is the template denaturation step. The template denaturation step requires high temperature of at least 90° C. In this step, DNA polymerase may be denatured. The DNA polymerases initially employed have low thermo-stability which is called mesophilic DNA polymerase. In this case with mesophilic DNA polymerase, fresh DNA polymerase has to be added to the PCR reaction mixture in each PCR cycle. However, since a thermo-stable DNA polymerase was found in Thermus aquaticus, a thermopile living in hot spring, the addition of fresh DNA polymerase to PCR reaction mixture in each PCR cycle has not been necessary and DNA polymerase is added just once when PCR is started. The optimal temperature for this kind of thermo-stable DNA polymerase (Taq DNA polymerase) is 72° C. and it is still stable at 94° C. The discovery of the thermo-stable Taq DNA polymerase facilitated PCR and paved a way for PCR to be used in various research fields (Science 252: 1643-1651, 1991). So now, PCR is acknowledged as a powerful technique used in various research fields.
Since the discovery of the thermo-stable Taq DNA polymerase, PCR techniques have been astonishingly advanced mainly by the discovery of novel DNA polymerases and the development of novel PCR techniques. Newly discovered or developed DNA polymerases are Tth DNA polymerase (from Thermus thermophilus), Tfl DNA polymerase (from Thermus flavus), Hot Tub DNA polymerase (from Thermus ubiquitos), Ultma DNA polymerase (from Thermotoga maritima), Pfu DNA polymerase (from Pyrococcus furiosus), Vent DNA polymerase (from Thermococcus litoralis) and Tli DNA polymerase (from Thermococcus litoralis) and Pwo DNA polymerase (from Pyrococcus woesei). Because these DNA polymerases are distinguished from one another in their characteristics, they have been utilized in different PCRs according to their unique properties. Precisely, they are different in DNA synthesizing speed, the number of nucleotides synthesized from the binding of the polymerase to a template DNA to the separation, preference to the kinds of template-primer, and sensitivity to inhibitory materials. Recently, a method has been developed to use at least two of these DNA polymerases together. Using this blend of different DNA polymerases is expected to have advantages because merits of both or multiple DNA polymerases can be all utilized or the overall inhibitory effect by an inhibitor can be reduced.
PCR techniques developed so far are as follows: rapid PCR characterized by reduced time for amplification; direct PCR capable of direct using of unpurified samples; reverse transcriptase-PCR (RT-PCR) which combines reverse transcription with PCR and thereby can use RNA molecule as a template; and real-time PCR facilitating real-time monitoring of PCR reaction. In addition, many techniques and methods have been developed but detailed explanations on these are not given herein.
In parallel with the development of new DNA polymerases and novel PCR techniques, studies have been undergoing to reduce “non-specific amplification” which is very a common problem encountered in general PCR. The major cause of non-specific amplification is that some primers in PCR reaction mixture anneal to templates before reaction temperature reaches desired and proper reaction temperature for PCR and then amplification by DNA polymerase is induced already to some degree. Besides, such non-specific amplification can also be significantly induced when an inappropriate primer not capable of securing the annealing between a template and a primer is used. Instructions have been given to design an appropriate primer which is well understood by those in the art, so that explanation is not necessary herein. There are other reasons for the non-specific amplification, for example inappropriate magnesium ion concentration in PCR reaction mixture, etc, but the major causes are the above two, so that minor causes are not explained herein.
As mentioned hereinbefore, in general PCR, a target sequence of a template is amplified by repeated PCR cycle of annealing and elongation after denaturation of the template DNA. The proper reaction temperature for PCR is generally higher (at least 40° C.) than room temperature (20-35° C. in general). But, as explained hereinabove, annealing between a primer and a template can happen at room temperature which leads to the amplification by DNA polymerase. Such amplification induced before reaching proper PCR temperature is based on non-specific template-primer annealing, so that it resultingly causes serious non-specific amplification. Annealing between a primer and a template happening at a less stringent annealing temperature lower than proper PCR reaction temperature is characterized by low specificity, so that the amplification based on such annealing with low specificity might include amplification of other non-target regions as well as a target region. The annealing between a template and a primer is generally determined by Tm of the used primer, which is also well known fact to those in the art, so that the additional explanation on that is not given here in this invention.
According to the conventional art, in order to reduce non-specific amplification at room temperature, a crucial component for PCR is not added to the PCR reaction mixture during the initial set-up stages of PCR and just prior to PCR cycling the component is added lastly (Nucleic Acids Res. 19: 3749, 1991). Magnesium ion has been selected as the omitted component in this conventional method. But, the conventional method does not facilitate the preparation of PCR reaction mixture at a time, causing inconvenience for experimenters.
Another example of the conventional art is that DNA polymerase is withheld physically, chemically, or biochemically not to participate in the amplification until temperature reaches desired and proper temperature for PCR. For this method, an antibody has been used (BioTechniques 16: 1134-1137, 1994). Or a chemical that is able to inactivate DNA polymerase by chemical modification has been used (the representative example of chemically modified DNA polymerase is AmpliTaq Gold DNA polymerase). Oligonucleotide binding to the active site of DNA polymerase has been also used (J. Mol. Biol. 264: 268-278, 1997). DNA polymerase physically, chemically, or biochemically arrested by foregoing materials is not functional at room temperature and once temperature reaches to denaturing temperature of a template during PCR, the arrested DNA polymerase is released and begins to work normally by the effect of the high temperature. As a result, the amplification at room temperature can be suppressed and accordingly hot-start PCR can be realized. Precisely, a chemical modifier that arrests DNA polymerase is degraded at template-denaturing temperature or an antibody or oligonucleotide is taken apart from DNA polymerase, so that DNA polymerase can work normally, suggesting that amplification by PCR is carried out after the denaturation stage of a template. This method has been quite effective so far, so that it has been widely used. However, this method has disadvantages of high costs and complication for its accomplishment.
It has been requested to develop a novel technique performed with less costs and with easy. To meet the request, it has been tried that amplification is suppressed until reaction temperature reaches to the proper temperature for PCR only by manipulating a primer. An example of the above trial is described in Korean Patent No. 649165. According to this description, a regulator was additionally inserted in the original primer. This regulator is polydeoxyinosine linker and inosine that composes the regulator is a universal base which has lower Tm than general nucleotides such as G, A, T and C. Therefore, polydeoxyinosine linker forms a bubble like structure at specific temperature to inhibit non-specific binding of a primer to a template, resulting in the inhibition of non-specific amplification of PCR. Compared with the said conventional arts, this method requires less cost for the accomplishment but a unique primer containing inosine is necessary, suggesting that this method is still inconvenient. Besides, annealing temperature (proper reaction temperature for PCR) of the first PCR cycle has to be different from that of the second PCR cycle, still causing inconvenience. The use of different annealing temperatures over the PCR cycle enables the additional sequence inserted into the original primer as well as the original primer sequence to participate in annealing between a template and a primer from the second PCR cycle. These different annealing temperatures over the PCR cycle are not always necessary but for the efficient PCR, the annealing temperature has to be switched over the PCR cycles. According to the above method, pre-selective arbitrary nucleotide sequence has to be added to 5′-terminus of primer but at this time, the pre-selective arbitrary nucleotide sequence is supposed not to be complementary to any of regions of target gene sequence, which makes the method more complicated and if the entire target gene sequence is not identified the success of this method will be in doubt. So, a novel method asking lower prices with easiness is needed.
The technique to reduce non-specific amplification is of course important for PCR, particularly for PCRs utilized in gene analysis or diagnosis of a disease.
The present inventors tried to develop a novel method which is simple and requires less costs. As a result, the inventors developed a PCR primer capable of inhibiting non-specific amplification by supporting both hot-start PCR and the amplification of PCR product rather than the amplification of original template, leading to the completion of this invention.
Numbers of papers and patent descriptions have been cited in this description and the citation is marked in parentheses. The descriptions of cited papers and patent documents are attached in this invention so that the art and text of this patent can be more clearly understood.